Project Summary Reversible epigenetic modifications on DNA and proteins play essential roles in controlling gene expression. Our laboratory recently demonstrated that RNA also contains reversible modifications and that they play essential roles in processes including fertility, development, and leukemia. Notably, modifications on RNA hold critical functions in the viral life cycle, affecting viruses such as Zika virus, HIV-1, and Hepatitis C virus. 2?-O-Methyl (2?OMe) is an abundant post-transcriptional RNA modification in eukaryotic and viral RNA, and recent studies showed that its presence on the 5? cap of viral RNA protects viruses from detection by the host immune response. We have identified that 2?OMe is also present on non-terminal nucleotides located internally in viral RNAs. However, the roles and locations of these internal 2?OMe marks remain undefined. Understanding how these 2?OMe marks affect viral infection may allow us to manipulate 2?OMe to suppress infection. To understand the roles of internal 2?OMe marks, we recently developed Nm-seq, a novel high-throughput sequencing technique that profiles 2?OMe at single-nucleotide precision with high sensitivity. This proposal aims to use Nm-seq and a combination of virological, molecular biological, biochemical, and genetic approaches to identify biological roles of internal 2?OMe in viral infection. Based on studies in other systems, we hypothesize that internal 2?OMe affects viral infection by impacting viral RNA replication, translation efficiency, interactions with host immune sensors, and infectious virus production. We will first use Nm-seq to determine the locations of internal 2?OMe modifications in two model viruses: Zika virus and Murine norovirus. We will then determine the functions of 2?OMe in the viral life cycle using RNA-dependent RNA polymerase replication assays of 2?OMe-modified or unmodified viral RNA; in vitro translation assays of 2?OMe-modified or unmodified viral RNA; RNA affinity chromatography followed by mass spectrometry to identify host proteins that bind preferentially to 2?OMe- modified or unmodified RNA; and viral infection experiments in cells depleted of a 2?OMe methyltransferase to measure infectious virus production. Our results will be the first to elucidate mechanisms by which internal 2?OMe affects viral infection, laying the groundwork for studies of therapeutic avenues to treat viral infection by targeting 2?OMe marks and the processes they affect. Our findings will also serve as a foundation for future investigations of 2?OMe in viral and eukaryotic RNA.